Ellipsometry system for measuring molecular binding, adsorption and desorption

ABSTRACT

According to one aspect of the invention, there is provided an ellipsometry system for measuring any one or more of molecular binding, adsorption and desorption on a substrate, the system comprising: a) a cuvette comprising i) a body within which a cavity is formed and an opening on the body, wherein the cavity extends into the opening through which the substrate is immersed; ii) a window formed on each of two oppositely located walls of the body, wherein the windows are aligned to allow light to enter through one of the two windows to reflect off the portion of the substrate immersed in the cavity and exit through the other of the two windows; iii) a channel arrangement enclosed within the body of the cuvette and comprising two non-contiguous portions, wherein one of the two non-contiguous portions guides fluid into the cavity and the other non-contiguous portion guides fluid out of the cavity; b) a polarized light source disposed to provide the light that enters into one of the two windows on the body of the cuvette; and c) a detection stage disposed to receive the light that exits through the other of the two windows on the body of the cuvette, wherein the detection stage is configured to measure polarization rotation of the received light, the polarization rotation caused by any one or more of molecular binding, adsorption and desorption occurring on the substrate surface. The detection stage preferably contains a polarisation modulator, which is configured to measure polarization rotation of the received light.

TECHNICAL FIELD

The present invention relates to an ellipsometry system for measuringany one or more of molecular binding, adsorption and desorption.

BACKGROUND ART

Ellipsometry is a technique which measures the change in polarization oflight when reflected off a sample surface. It derives its sensitivityfrom the determination of the relative phase shift Δ between normal (p)and in-plane (s) components of polarization vector in the reflectedlight. The phase shift can be measured with precision of 0.1 degree,which translates to around 1 nm of organic material on silicon substratein air.

Precision ellipsometry (PREL) is based on modulation of polarization,which further increases sensitivity to 0.01 mrad, which translates to0.01 nm. This high sensitivity allows real time measurement of atomicand molecular attachment and detachment.

FIG. 1 shows a prior art system that is used for precision ellipsometry.Not shown are a light source, which produces linearly polarized lightthat is made incident 104 on a substrate 106 providing the samplesurface, and a polarization analyser and detector 108 placed on the pathof the reflected beam 110.

A cuvette 120 can be used to introduce a layer, where any one or more ofmolecular binding, adsorption and desorption occurs, onto the substrate106 surface. The cuvette 120 is of closed flow type and has an inlet 122and an outlet 124 only for liquid. The substrate 106 must be placedwithin the cuvette 120 in advance before closing and optical alignment.While the closed flow cuvette 120 allows quick exchange of solvent andsolution, it requires a long time to change the substrate 106, becausethis involves draining of liquid before opening draining of air afterclosing and alignment of the optical analysis system (not shown) foreach substrate 106.

FIG. 2 shows a prior art immersion cuvette 220. The immersion cuvette220 of FIG. 2 has an inlet 222 and an outlet 224 for liquid and anopening 226 through which a substrate 206 can be immersed. The advantageof this cuvette 220 is the possibility of fast changing the substrate206 together with keeping all other parts of the system stable. Thuswhen a next substrate (not shown) is immersed, the system is ready forthe next measurement. However liquid flow pattern is complicated becausethe inlet 222 and the outlet 224 are introduced from the top of thecuvette 220. Hence a magnetic stirrer 227 is required to achieveuniformity of the solution, and hence it is suitable to measure onlyslow kinetics, e.g. in the range of minutes.

Another optical technique for measurement of molecular binding issurface plasmon resonance (SPR), which is shown in FIG. 3. SPR relies ona measurement of refractive index change at a metal surface, whenmolecules attach to the surface. It works as follows: light 304 entersthrough a transparent substrate 306 at incidence angle θ_(SPR). Thelight 304 induces plasmon resonance in a gold (Au) layer 315; depositionof the layer 313 changes the value of the resonance angle. If incidenceangle θ_(SPR) is fixed, it changes reflected intensity. Because surfaceplasmon wave is evanescent in the direction normal to the surface andpropagates along the surface, it is very sensitive to the molecules nearthe surface and less sensitive to molecules far from the surface, orchanges in the medium. However, SPR only works with noble metals (mostlygold), which limits the range of applications of this measurementtechnique.

There is thus a need to address the shortfalls of the measurementtechniques described above.

SUMMARY OF INVENTION

According to one aspect of the invention, there is provided anellipsometry system for measuring any one or more of molecular binding,adsorption and desorption on a substrate, the system comprising: a) acuvette comprising i) a body within which a cavity is formed and anopening on the body, wherein the cavity extends into the opening throughwhich the substrate is immersed; ii) a window formed on each of twooppositely located walls of the body, wherein the windows are aligned toallow light to enter through one of the two windows to reflect off theportion of the substrate immersed in the cavity and exit through theother of the two windows; iii) a channel arrangement enclosed within thebody of the cuvette and comprising two non-contiguous portions, whereinone of the two non-contiguous portions guides fluid into the cavity andthe other non-contiguous portion guides fluid out of the cavity; b) apolarized light source disposed to provide the light that enters intoone of the two windows on the body of the cuvette; and c) a detectionstage disposed to receive the light that exits through the other of thetwo windows on the body of the cuvette, wherein the detection stage isconfigured to measure polarization rotation of the received light, thepolarization rotation caused by any one or more of molecular binding,adsorption and desorption occurring on the substrate surface. Thedetection stage preferably contains a polarisation modulator, which isconfigured to measure polarization rotation of the received light.

BRIEF DESCRIPTION OF DRAWINGS

Example embodiments of the invention will be better understood andreadily apparent to one of ordinary skill in the art from the followingwritten description, by way of example only, and in conjunction with thedrawings. The drawings are not necessarily to scale, emphasis insteadgenerally being placed upon illustrating the principles of theinvention, in which:

FIG. 1 shows a prior art closed flow through cuvette.

FIG. 2 shows a prior art immersion cuvette.

FIG. 3 shows prior art measurement of molecular binding using surfaceplasmon resonance.

FIG. 4A shows a schematic of a system, in accordance with one embodimentof the present invention, for precision ellipsometry to measure any oneor more of molecular binding, adsorption and desorption.

FIG. 4B shows measurement of a polarization signal received beforemolecular adsorption takes place in the system of FIG. 4A.

FIG. 4C shows measurement of a polarization signal received aftermolecular adsorption has occurred in the system of FIG. 4A.

FIG. 4D shows a polarization signal received after retardation in thesystem of FIG. 4A.

FIG. 5A shows a front view of a cuvette made in accordance with oneembodiment of the present invention.

FIG. 5B shows a side view of the cuvette of FIG. 5A.

FIG. 5C shows a top view of the cuvette of FIG. 5A.

FIG. 6 shows the ellipsometry system, in accordance with one embodimentof the invention, comprising a pump.

FIG. 7A shows a graph which plots the thickness of adsorption when usingeach of various solvents.

FIG. 7B shows measurement of polarization rotation that results from 1nm of organic layer on a silicon substrate in air and in water.

FIG. 8A shows a side view of fluid flow in the cuvette shown in FIGS. 5Ato 5C.

FIG. 8B shows a front view of fluid flow in the cuvette shown in FIGS.5A to 5C.

FIG. 9A shows a plot of flow-through fraction against time obtained fromusing the cuvette shown in FIGS. 5A to 5C.

FIG. 9B shows the results obtained from real-time measurement of surfacebinding using the cuvette shown in FIGS. 5A to 5C.

FIG. 10A shows a functional schematic of a detection stage in accordancewith one embodiment of the present invention.

FIG. 10B shows a schematic of a first implementation of the drivemechanism shown in FIG. 10A.

FIG. 10C shows a schematic of implementation of polarisation modulatorwith polarizer suspended on several springs.

FIG. 10D shows a front view of a schematic of a second implementation ofthe drive mechanism shown in FIG. 10A.

FIG. 10E shows a front view of a schematic of a third implementation ofthe drive mechanism shown in FIG. 10A.

FIG. 10F shows a side view of a schematic of the third implementation ofthe drive mechanism shown in FIG. 10E.

FIG. 11 shows a plot of the dependence of light intensity through themodulator against angle between polarization from using the thirdimplementation of the drive mechanism shown in FIGS. 10E and 10F.

FIG. 12A shows results of using the detection stage of FIG. 10A to studymagnetic materials having magneto-optical Kerr effect.

FIG. 12B shows a schematic where the detection stage of FIG. 10A is usedfor remote detection of magnetic field, where wired measurement is notavailable.

FIG. 13 shows the results of using the detection stage of FIG. 10A tomeasure rotation of polarisation of light passing through a 3 mm vesselwith water and after adding sugar solution in various concentrationsteps.

FIG. 14A shows the results of using the detection stage of FIG. 10Ausing the setup shown in FIGS. 14B and 14C.

FIG. 14B shows instantaneous monitoring of sugar and flow duringmeasurement.

FIG. 14C shows flow at the time of rinsing and calibration

DETAILED DESCRIPTION

In the following description, various embodiments are described withreference to the drawings, where like reference characters generallyrefer to the same parts throughout the different views.

FIG. 4A shows a schematic of a system 400, in accordance with oneembodiment of the present invention, for precision ellipsometry tomeasure any one or more of molecular binding, adsorption and desorption.The system 400 comprises a light source 402, such as a laser, having apolarizer 405 that produces linearly polarized light which is madeincident 404 on a sample 406. A polarization analyser and detector 408are placed on the path of the reflected beam 110.

At an arbitrary incident angle when the sample 406 does not have a layer413, both the incident ray 404 and the reflected ray 410 are linearlypolarized, as shown in FIG. 4B. Deposition of the layer 413 changespolarisation of the reflected light 410 from linear to elliptic, asshown in FIG. 4C, where the phase shift Δ is proportional to thethickness d of the layer 413, as set out in equation (1) below, wherethe thickness d is less than wavelength of the polarised light. Thepolarisation analyser and detector 408 measures the ellipticity Δ inaccordance with equation (2) below which is based on a photo-elasticmodulator (PEM) or a Faraday rotation cell.

$\begin{matrix}\left\lbrack {{Math}.\mspace{14mu} 1} \right\rbrack & \; \\{\Delta = {2\beta \frac{{r_{1}^{p}r_{2}^{s}} - {r_{2}^{p}r_{1}^{s}}}{\tan \; {\Psi \left( {r_{1}^{s} + r_{2}^{s}} \right)}^{2}}}} & (1) \\\left\lbrack {{Math}.\mspace{14mu} 2} \right\rbrack & \; \\{\beta = {\frac{2\pi \; d}{\lambda}\sqrt{n_{1}^{2} - {n_{0}^{2}\sin^{2}\theta_{1}^{i}}}}} & (2)\end{matrix}$

To measure the ellipticity of the reflected light, a quarter-waveretarder 414 is placed before the polarisation analyser 419 and detector408. The retarder 414 converts ellipticity Δ into rotation γ, as shownin FIG. 4D, which is measured using the polarisation analyser 419 anddetector 408. The rotation γ is proportional to the thickness d inaccordance with equation (3) below.

γ=Δ sin Ψ cos Ψ  (3)

where Ψ is angle of long axis of ellipse and Δ is phase shift betweennormal (p) and in-plane (s) components of reflected polarization vectorshown in FIG. 1C

FIGS. 5A to 5C show a front view, a side view and a top viewrespectively of a cuvette 520 made in accordance with one embodiment ofthe present invention. FIG. 5B is a side view of a partial cross-sectionof the cuvette 520, with a portion cut away to show the internalcomponents of the cuvette 520. The features of the cuvette 520 aredescribed with reference to FIGS. 5A to 5C.

With reference to FIG. 5A, the cuvette 520 comprises a body 522 withinwhich a cavity 524 (see FIG. 5B) is formed and an opening 526 on thebody 522. The cavity 524 is an empty space contained within the depth ofthe body 522 (i.e. the cavity 524 is not a through hole) and is forstoring fluid. The cavity 524 extends into the opening 526, so that thecavity 524 is accessible via the opening 526.

The body 522 comprises a support structure 528. The support structure528 is adapted to hold the substrate 506 with at least a portionimmersed in the cavity 524, i.e. the support structure 528 immerses atleast a portion of a substrate 506 within the cavity 524. The supportstructure 528 thus provides the body 522 with a dedicated part that isdesignated to hold the substrate 506. During use, as shown in FIGS. 5Ato 5C, the substrate 506 is inserted through the opening 526 to beplaced on the receiving surface 556 of the support structure 528 withinthe cavity 524. In the embodiment shown in FIGS. 5A to 5C, at least theportion of the support structure 528 against which the substrate 506lies is integral with the body 522 of the cuvette 520, i.e. that portionis unitary with the body 522. In another embodiment (not shown), theentire support structure 528 is manufactured separately from the body522 and then affixed to the body 522.

A fluid inlet 532 and a fluid outlet 534 are formed on the body 522. Achannel arrangement 536 is enclosed within the body 522 of the cuvette520. The channel arrangement 536 comprises two non-contiguous portions,with each non-contiguous portion extending into the cavity 524. One ofthe two non-contiguous portions guides fluid into the cavity 524 and theother non-contiguous portion guides fluid out of the cavity 524. Thefluid inlet 532 and the fluid outlet 534 are each in fluid communicationwith the channel arrangement 536. The enclosed channel arrangement 536provides an input conduit 538 between the fluid inlet 532 and the cavity524, and an output conduit 540 between the cavity 524 and the fluidoutlet 534. The input conduit 538 provides one of the two non-contiguousportions of the channel arrangement 536, while the output conduit 540provides the other of the two non-contiguous portions of the channelarrangement 536. Since the cavity 524 is where the substrate 506 islocated, the end of the input conduit 538 which is connected to thecavity 524 becomes the region where fluid that enters the fluid inlet532 is first introduced to the substrate 506, while the end of theoutput conduit 540 which is connected to the cavity 524 becomes theregion where this fluid is extracted from the cavity 524. Thus theenclosed channel arrangement 536 provides a means to control the fluidflow line pattern experienced by the substrate 506. Compared to simplyproviding the inlet 222 and the outlet 224 through the opening 226 ofthe cuvette 220 shown in FIG. 2, the enclosed channel arrangement 536thus improves control over the fluid flow line pattern experienced by asubstrate. The embodiment shown in FIGS. 5A to 5C has the channelarrangement 536 implemented as two separate conduits 538 and 540, witheach provided in a different wall or part of the body 522. However, itwill be appreciated that other configurations (not shown) may have thechannel arrangement 536 disposed within a same part or wall of the body522. Further, the part of the body 522 where the channel arrangement 536is located can be varied, so that desired flow lines can be achieved.

The body 522 has several walls 542 with each wall providing a planarsurface. A window 544 is formed on each of two oppositely located walls542 a and 542 b of the body 522. These walls 542 a and 542 b areoppositely located in that the walls 542 and 542 b are located on othersides of the body 522. The windows 544 are aligned to allow light toenter 504 through one of the two windows 544 to reflect off the portionof the substrate 506 suspended within the cavity 524 and exit 510through the other of the two windows 544. Thus, the provision of thesupport structure 528 ensures that any substrate 506 is placed in aposition to reflect the light from one of the two windows 544 to theother of the two windows 544. Such an alignment reduces the need toperform optical realignment when the substrate 506 is changed, since thesupport structure 528 ensures that the substrate 506 will be placed inthe same position. Achievement of this alignment may thus be consideredduring determination of the location of the support structure 528 withinthe body 522 of the cuvette 520.

The cuvette 520 is a component of an ellipsometry system 500 that isshown in FIGS. 5A and 5C. The ellipsometry system 500 includes apolarized light source 502 and a detection stage 508. The polarizedlight source 502 is disposed to provide the light 504 that enters intoone of the two windows 544 on the body 522 of the cuvette 520. Thedetection stage 508 is disposed to receive the light 510 that exitsthrough the other of the two windows 544 on the body 522 of the cuvette520. The detection stage 508 is configured to measure polarizationrotation of the received light 510, wherein the polarization rotation iscaused by any one or more of molecular binding, adsorption anddesorption occurring on the substrate 506 surface.

The channel arrangement 536 comprises the input conduit 538 that iscoupled at one end to the fluid inlet 532 and coupled to the cavity 524at the other end. As shown in FIG. 5B, the input conduit 538 extendsinto the cavity 524 by going around a segment 558 of the portion of thesupport structure 528 suspended within the cavity 524. This segment 558of the support structure 528 is, in one embodiment, located at thebottom of the support structure 528, the bottom being located within thecavity 524.

In the embodiment shown in FIGS. 5A to 5C, the input conduit 538comprises two branches 546, wherein each of the two branches 546 has oneend that meets at a common region 548 for coupling to the fluid inlet532 and the other end is coupled to opposite regions 552 of the cavity524 relative to the portion of the support structure 528 suspendedwithin the cavity 524. In one embodiment, these opposite regions 552 arelocated at opposite corners of the cavity 524, whereby such a locationensures that fluid flow does not have stagnant corners. In analternative configuration (not shown), the input conduit 538 comprisesof only a single branch which directly connects the fluid inlet to thecavity.

The channel arrangement 536 also comprises the output conduit 540 thatis coupled at one end to the fluid outlet 534 and coupled to the cavity524 at the other end. Unlike the input conduit 538 of the embodimentshown in FIGS. 5A to 5C, the output conduit 540 is a singular branch.

The support structure 528 has a surface 556 that receives the substrate506 and an opposite surface that faces the input conduit 538 of thechannel arrangement 536. This receiving surface 556 of the supportstructure 528 is the surface against which the substrate 506 rests. Inaddition, the support structure 528 has a biasing element 554 that urgesthe substrate 506 against the receiving surface 556.

Although the biasing element 554 used in the embodiment shown in FIGS.5A to 5C is a leaf affixed on one end to the body 522 of the cuvette520, with the other end used to apply pressure on the substrate 506, thebiasing element 554 may also any one or more of a screw and a clip,although these alternative configurations are not shown.

The body 522 of the cuvette 520 further comprises a reagent inlet 550that extends into the channel arrangement 536 which is in fluidcommunication with the fluid inlet 532. In the embodiment shown in FIGS.5A to 5C, the reagent inlet 550 thus extends into the input conduit 538.The reagent inlet 550 provides a port into which reagent is introducedinto the cavity 524, whereby the reagent is transported to the cavity524 via fluid introduced into the cuvette 520 through the fluid inlet532. The reagent inlet 550 is on the same surface of the body 522 wherethe opening 526, into which the cavity 524 extends, is located. Theopening 526, the fluid inlet 532 and the fluid outlet 534 are located ondifferent surfaces of the body 522 of the cuvette 520.

The ellipsometry system 500 may further comprise a pump 660 coupled tothe fluid outlet 534 of the body 522 of the cuvette 520, as shown inFIG. 6. The pump 660 may be a peristaltic pump, which can pump eitherair or liquid. The pumping rate may be set higher than the inlet flowrate, hence the constant pumping keeps the liquid level constant with orwithout liquid inlet flow into the fluid inlet 532. If reagent orsolvent is injected into the cuvette 520 faster than the pumping rate,they will overflow. To prevent this, the volume above the outlet 534 isa reserve for in case reagents are too injected too quickly. Furtherfluidic accessories that the ellipsometry system 500 may have are asolvent source vessel 762 which is coupled to the body 522 via the fluidinlet 532 of the cuvette 520 via a flow control valve 764. The pump 660may also be coupled to a drain vessel 766.

Returning to FIGS. 5A to 5C, the cuvette 520 holds the substrate 506 inorder to measure molecular adsorption of reagent that is transported tothe substrate 506 by the fluid that that is injected into the fluidinlet 532. The cavity 524 of the cuvette 520 provides a reservoir thatcan contain a sufficient quantity of fluid to submerge the portion ofthe substrate 506 where any one or more of molecular binding, adsorptionand desorption is being observed.

Each of the two windows 544 is orientated to allow polarised light totransmit through perpendicularly; so that any molecules deposited on thewindows 544 do not affect light polarization. In order to examine anypossible interference of glass on measurement of molecular adsorption(i.e. in considering the impact of using glass to fabricate each of thetwo windows 544), a gas chamber with glass windows was made (not shown)and experiments using the vapour of propanol, ethanol, methanol andwater confirmed that adsorption of molecules on the substrate could bereproducibly measured. FIG. 7A shows a graph which plots the thicknessof adsorption when using each of these vapours. The requirement of glasswindows to be perpendicular to the light applies for molecular layers inliquid, because the value of polarization rotation will be smaller thanthose of vapour. FIG. 7B shows polarization rotation that results from 1nm of organic layer on a silicon substrate in air (curve 701) and inwater (curve 703).

The cuvette 520 is able to secure the substrate through the supportstructure 528 and allows substrate exchange as well as light beamadjustment. The fluid inlet 532 and the fluid outlet 534 allow fluid,such as solvent, to pass in and out of the cuvette 520. The reagentinlet 550 allows quick injection of reagent, such as solutions ofmolecules intended for interaction with the substrate 506, where thefluid outlet 534 allows the reagent to be removed via the fluidintroduced through the fluid inlet 532.

The body 522 of the cuvette 520 may be made of, for example, acrylicslab, plastic, metal, glass or ceramic. The substrate 506 may be of madefrom any material, such as plastic, insulator or semiconductor, with areflecting surface. The fluid that is introduced through the fluid inlet532 may be aqueous solution, inorganic salt solution or solutions inorganic solvents. Reagent that is introduced into the reagent inlet 550may include any one or more of organic molecules, polymers andbiomolecules. The biomolecules may include any one or more of protein,nucleic acids, sugar, peptides, enzyme, cell and cell membrane.

The cavity 524 provides for a fluidic reservoir in front of thesubstrate 506. In the embodiment shown in FIGS. 5A to 5C, the cavity 524has the shape of a vertical trapezoidal prism having slanted sidesdefined by the opposing walls 542 a and 542 b. In another embodiment(not shown), the cavity may have a triangular shape. Each of theopposing walls 542 a and 542 b has an optical window 544 made ofinorganic transparent material, for example, glass or quartz. In orderto provide incoming light 504 at an angle θ to the substrate 506 normal,the window 544 is preferably slanted at the same angle θ with respect tothe substrate 506, so that the incoming light 504 is perpendicular tothe plane of the window 544 of the wall 542 a. Similarly, the reflectedlight 510 is also perpendicular to the plane of the window 544 of thewall 542 b. The substrate 506 is held using the biasing element 554against the rigid back wall of the cavity 524, the rigid back wall beingprovided by the support structure 528. This allows any substrate to beimmersed from the top of the cuvette 520 and stand rigidly at the sameposition, which keeps light beam adjustment.

The input conduit 538 that is located between the support structure 528and a rear wall 542 c (see FIG. 58) of the body 522 of the cuvette 520provides a fluidic channel that allows introduction of fluid (via thefluid inlet 532), such as liquid, and reagent (via the reagent inlet550), such as solution, into the opposite regions 552 located at thebase of the cavity 524. In the embodiment shown in FIGS. 5A to 5C, theseopposite regions 552 are located at the bottom corners of the cavity524. In an alternative configuration (not shown), the fluidic channelprovided by the input conduit does not connect to the base of thecavity, but may connect to the cavity at, for example, between the baseof the cavity and the height at which the fluid outlet 534 is located.The fluid outlet 534 is located around the middle height of the body522, away from the path of the incoming and outgoing light 504 and 510.With reference to FIG. 7C, the design of the cuvette 520 allows fluidflow lines 719, within the cavity 524, that start from the inlets 752 ofthe cavity 524 (corresponding to the opposite regions 552 shown in FIGS.5A and 5C) and end at the outlet 734 of the cavity 524, the outlet 734leading to the fluid outlet 534 shown in FIGS. 5A to 5C. It will beappreciated that there are no standing corners; the bottom corners (i.e.the opposite regions 552) are inlets to the cavity 524, while the topcorner is formed by meniscus 717 of the fluid in the cavity 524, i.e. afree surface.

FIGS. 8A and 8B respectively show a side view 801 and front view 803 offluid flow in the cuvette 520, shown in FIGS. 5A to 5C, as studied usingcomputational fluid dynamics simulation. The two inlets 752 (shown inFIG. 7C) located at the bottom of the cavity 524 are represented usingreference numeral 805, while reference numeral 807 represents the outlet734 located at the top of the cavity 524. The flow pattern is shown bythe particle path lines 819. The liquid sample injected into the cuvette520 via the two inlets 805 flow upward leaving the cuvette 520 via theoutlet 807. Most of the liquid passes through the centre of the cuvette520 where the substrate 506 is attached and the dead corner isrelatively insignificant.

The cuvette 520 shown in FIGS. 5A to 5C has several applications whichinclude real time study of chemical reactions, where one reagent isimmobilised on a reflecting substrate and the other is supplied from thesolution; heterogeneous catalysis; monitoring of production in chemicaltechnology; imaging, if equipped with an array detector, quantitativeand qualitative analysis of binding of biological solutions andsuspensions; and studying fouling and anti-fouling processes.

FIG. 9A shows a plot 900 of flow-through fraction against time of thecuvette 520 shown in FIGS. 5A to 5C. The design of the cuvette 520allows quick solution exchange. It only needs less than 10 seconds toreplace half of the solution, which is mostly in the centre of thecuvette 520. More than 90% of the liquid is replaced with fresh liquidin less than 30 seconds. For the plot 900, the liquid is injected at0.01 m/s. Shorter replacement duration is expected when a higher inletvelocity is used. These results also show that the quick removal of thesolution in the cuvette is comparable to a “flow-through” cuvette.

The procedure for specific molecular binding measurements using thecuvette 520 shown in FIGS. 5A to 5C is carried out by immobilising areceptor molecule on the substrate 506 and applying a target molecule ina solution. This is so called heterogeneous or solid-liquid phasedetection. The substrate 506, cleaned using wet chemistry, is immersedinto the cuvette 520 filled with liquid injected into the cuvette 520through its fluid inlet 532. This allows molecules inside the injectedliquid to immobilise on the substrate 506 surface. The optical system,provided by the polarized light source 502 and the detection stage 508is adjusted to get a first polarization signal reading. Then liquid ispassed through the cuvette 520 to obtain a stable environment andoptical signal recording is started. At a certain moment, the flow ofliquid is stopped and a respective reagent (i.e. the receptor or thetarget) is injected into the reagent inlet 550, while excess of liquidis removed through the fluid outlet 534 by the pump 660 (see FIG. 6). Asecond polarization signal reading is then obtained. If the reagentbinds to the surface of the substrate 506, then polarization rotationincreases showing the increase of thickness of a layer formed by thereagent bound to the surface of the substrate 506. However, if there isa decrease of rotation, it means removal of molecules from the surfaceof the substrate 506. The difference between the polarization rotationof the first reading and the second reading is proportional to averagethickness of a layer formed by the reagent on the substrate 506 surface.

If the solutions (i.e. both the liquid injected through the fluid inlet532 and the reagent injected through the reagent inlet 550) used aretransparent, measurement of the binding can be done in real time,because the rotation of polarization can be continuously recorded. Thereal-time measurement allows for kinetic analysis for specific surfacebinding process. The design of the cuvette, which enables fast change ofsolvent to solution and vice versa, allows measurements of kinetics asfast as in seconds.

If the solutions used are not transparent, the ellipsometry system 500still enables quantitative measurement of the amount of molecules thatbind to the surface of the substrate 506 by comparing signal at rinsinglevel before injection of the solution and after removal of thesolution. The difference between these levels can be converted to theamount of attached material (although kinetics may be not visible). Thismethod of measurement by comparing rinsing levels is useful for analysisof biological solutions and suspensions.

FIG. 9B shows the results obtained from real-time measurement of surfacebinding using the cuvette 520 shown in FIGS. 5A to 5C. Firstly asolution of aminosilane is injected into the fluid inlet 532. Theaminosilane binds to an oxidised Si sample used for the substrate 506through formation of covalent bonds. At higher concentration, thebinding is faster, while after rinsing, the final thickness is the same:around 1 nm, indicating irreversible attachment of one molecular layer.Subsequently solution of poly-styrene-sulfonate (PSS) is injected intothe reagent inlet 550. The negatively charged PSS binds to the surfaceof the substrate 506 through electrostatic interaction with thepositively charged amino-groups —NH3+. Here the concentration affectsnot only attachment rate, but also the final thickness, because polymerscan attach to surfaces in different conformations. In this example,aminosilane is the receptor and PSS is the target. It is seen thatattachments of both aminosilane and PSS are irreversible, i.e. the flowof water does not remove the molecules. It is seen also that kinetics ofattachment depends differently on concentration, indicating differentprocesses during attachment of small molecules and polymers.

The cuvette 520 shown in FIGS. 5A to 5C allows for real-time monitoringof the rate and progress of chemical reactions, as opposed to methodswhich extract samples of the reaction mixture at various period of timefor analysis to determine the amount of product and precursors presentat each point of time. Such methods are troublesome and complicated andare incapable of detecting the rate and progress as the reactionproceeds. Unlike other real-time monitoring systems which use expensiveand complicated equipment like nuclear magnetic resonance and requiretechnical expertise, the cuvette 520 provides a cost effective apparatusto facilitate real-time monitoring.

There are existing ellipsometry systems that use the same underlyingprinciple employed by the cuvette 520 shown in FIGS. 5A to 5C, i.e.molecular interaction where a precursors (or receptor) is immobilised ona substrate, while a target molecule is introduced in the surroundingmedium, followed by measurement of the attachment, detachment, orreaction between the molecules. In monitoring changes in thickness of alayer on the substrate, standard ellipsometry has a precision of around1 nm in gas and vacuum, and around 10 nm in liquid. To monitor molecularlayers in the order of a nanometer, there are several techniques basedon special substrates, such as surface plasmon resonance (SPR) describedwith reference to FIG. 3. SPR requires a gold layer 315 on the substrate306. Quartz crystal microbalance (QCM) requires quartz crystal substratewith electrodes, while dual polarisation interferometry (DPI) requirestwo waveguides in the substrate. All these substrates are more expensivethan those used for ellipsometry. Further, SPR only works with noblemetal (mostly gold) surface; QCM uses metal (gold, silver, copper) andmetal coated with a thin layer of metal oxide (TiO₂ or SiO₂). DPI islimited to silicon nitride.

The cuvette 520 shown in FIGS. 5A to 5C is advantageous over SPR becauseit works on the principle of ellipsometry and can use any substrate(such as plastic, insulator, or semiconductor) with a reflectingsurface. The cuvette 520 shown in FIGS. 5A to 5C enables measurement ofthe kinetics of surface binding process with high precision and at lowprice. Cost reduction is possible by use of inexpensive reflectingsubstrates and a polarization modulator, such as the one described ingreater detail with reference to FIGS. 10A to 10F. The polarizationmodulator described with reference to FIGS. 10A to 10F provides highprecision of ellipsometry measurement (down to 0.1 nm of organicmolecules in water).

FIG. 10A shows a functional schematic of the detection stage 508 shownin FIGS. 5A and 5C. The detection stage 508 does not necessarily have tobe used in tandem with the cuvette 520 of FIGS. 5A to 5C, since thedetection stage 508 is configured to measure rotation which polarizedlight, generated from a source, may experience after modulation. Suchpolarization rotation may occur, for instance, when polarized light isreflected from molecular adsorption occurring on a substrate surface.

The detection stage 508 comprises a modulator stage 1002 and a detector1006. The modulator stage 1002 and the detector 1006 are disposed alongthe path of the light 510 that is transmitted from the window 544 on thewall 542 b of the cuvette 520 (see FIG. 5A). A function generator 1007and a phase-lock amplifier 1005 are electrically coupled to thedetection stage 508. The function generator 1007 is electrically coupledto the modulator stage 1002 and the phase-lock amplifier 1005 iselectrically coupled to the detector 1006. The function generator 1007is electrically coupled to the phase-lock amplifier 1005. Use ofphase-lock detection of light intensity allows measurement of rotationof polarization of the light 510 with precision in the order ofmicroradian.

The modulator stage 1002 comprises a retarder 1014 and a polarizer 1012.The retarder 1014 is disposed in the path of the received light 510 toconvert the state of polarization of the received light 510 into apolarization suitable for the polarizer 1012. The polarizer 1012 isdisposed to receive light 510′ transmitted through the retarder 1014,the transmitted light 510′ having been converted into a polarizationstate suitable for the polarizer 1012 by the retarder 1014. Thepolarizer 1012 may be a polaroid or a dichroic sheet. The modulatorstage 1002 further comprises a drive mechanism coupled to the polarizer1012. The drive mechanism is coupled to the polarizer 1012 to modulatethe polarization of the light transmitted through the retarder 1014(i.e. the light 510′). The drive mechanism is configured to move thepolarizer within a plane generally perpendicular to the direction of thereceived light 510. The detector 1006 is disposed downstream of thepolarizer 1014 to receive light transmitted through the polarizer 1014.Accordingly, the polarizer 1012 is located between the retarder 1014 andthe detector 1006.

The movement that the drive mechanism causes the polarizer 1012 toundertake is over an angle range which is similar to the polarizationrotation that the light 510′ (i.e. light transmitted through theretarder 1014) has compared to the light 504 that enters the window 544on the wall 542 a of the cuvette 520 (refer FIG. 5A). Measurement of themovement that the drive mechanism makes would then be reflective of thepolarization rotation that has occurred. The drive mechanism iselaborated below with reference to FIGS. 10B to 10F. In FIGS. 10B to10F, the retarder 1014 is omitted for the sake of simplicity.

FIG. 10B shows a schematic of a first implementation of the drivemechanism described with reference to FIG. 10A. The drive mechanismcomprises an actuator 1015 coupled to the polarizer 1012, wherein theactuator 1015 is configured to move or slant the polarizer 1015 along anarc 1009 that lies in the plane 1017, the plane 1017 being generallyperpendicular to the direction of the light 510′ (denoted with the cross“x”) transmitted through the retarder 1014. This plane 1017 is alsogenerally perpendicular to the direction of the received light 510,since the transmitted light 510′ results from the light 510 received bythe retarder 1014 and is along the same path as the received light 510.The arrows 1003 show the direction of polarization of the polarizer1012. While the embodiment shown in FIG. 10B has the actuator 1015coupled to the polarizer 1012 through an elastic element 1019, such as aspring, another configuration (not shown) may have the actuator directlycoupled to the polarizer. The actuator 1015 is coupled to a shelf 1021.

FIG. 10B uses bold lines to depict the actuator 1015, the polarizer 1012and the spring 1019 at rest. Dashed lines are used to depict theactuator 1015, the polarizer 1012 and the spring 1019 when the actuator1015 is activated, bringing about the arc movement 1009. When theactuator 1015 is realised using a piezo-electric actuator, thepiezo-electric actuator is activated by applying an oscillating voltage.The piezoelectric actuator then bends and induces bending of the spring1019, which may be an elastic element or an elastic strip, and tilts thepolarizer 1012 to trace the arcuate path 1009. The tilt of the polarizer1012 produces an oscillating tilt of polarization of the light 510transmitting through the modulator stage 1002 (refer FIG. 10A). Maximumtilt is achieved, when frequency of the applied voltage coincides withresonant frequency determined by the mass of the polarizer 1012 andstiffness of the spring 1019. To decrease the operating voltage and/orsize of the piezoelectric actuator, the frequency of the applied voltagemay be set to coincide with the resonant frequency determined by themass of the polarizer 1012 and stiffness of the spring 1019. In theembodiment shown in FIG. 10B, the actuator 1015 is a piezo-electricstrip. In another embodiment (not shown), the actuator may be abimetallic strip.

FIG. 10C shows a front view a schematic of a second implementation ofthe drive mechanism described with reference to FIG. 10A. The drivemechanism comprises a platform 1029 coupled to the polarizer 1012,wherein the platform 1029 is arranged to rotate around an axis 1031 thatis generally parallel to the path of the received light 510, so that therotating platform 1029 is configured to rotate the polarizer 1021 on theplane that is generally perpendicular to the direction of the receivedlight 510. The rotating platform 1029 may have the actuator 1015, whichis coupled to the polarizer 1012 via the spring 1019. Similar to theembodiment shown in FIG. 10B, the actuator 1015 may be a piezo-electricstrip or a bimetallic strip. The rotating platform 1029 has an opening1035 over which the polarizer 1012 is suspended, the opening 1035allowing the light 510′ to pass through to the detector 1006 (see FIG.10A).

FIGS. 10D and 10E respectively show a front view and a side view of aschematic of a third implementation of the drive mechanism describedwith reference to FIG. 10A. The third implementation combines theplatform 1029 of FIG. 10C with the drive mechanism shown in FIG. 10B.The drive mechanism of the third implementation comprises the platform1029 coupled to the polarizer 1012, wherein the platform 1029 isarranged to rotate around an axis 1031 that is generally parallel to thepath of the light 510′ transmitted through the retarder 1014, so thatthe rotating platform 1029 is configured to rotate the polarizer 1012 onthe plane that is generally perpendicular to the direction of the light510′. The rotating platform 1029 has an opening 1035 over which thepolarizer 1012 is suspended, the opening 1035 allowing the receivedlight 510′ to pass through to the detector 1006 (see FIG. 10A). Theplatform 1029 is coupled to the polarizer 1012 through the shelf 1021mounted on the platform 1029, wherein the shelf 1021 has the actuator1015 that is coupled to the polarizer 1012 through the spring 1019. Asdescribed with reference to FIG. 10B, the actuator 1015 allows thepolarizer 1015 to move along an arc 1009 that lies in the planegenerally perpendicular to the direction of the light 510′. Thepolarizer 1012, the shelf 1021, the spring 1019 and the actuator 1015are housed within a protection box 1037 mounted on the platform 1029,wherein the shelf 1021 is coupled to the polarizer 1012 through thespring 1019

The rotating platform 1029 provides a rotation stage to which theactuator 1015 is attached. The rotating platform 1029 allows setting themean direction of polarization. While polarization modulation ispreferably performed by the arcuate motion brought about by the actuator1015 coupled to the polarizer 1015, to analyse the polarization rotationof the received light 510, the polarization modulation can bealternatively undertaken by the rotation stage provided by the rotatingplatform 1029. In this alternative undertaking, the rotation stage setsthe mean direction to extinction and the light that passes through thepolarizer 1015 depends on the rotation of polarization of the incominglight 510.

In an alternative configuration shown in FIG. 10F, the polarizer 1012 issuspended using a plurality of spatially arranged elastic elements 1019.In the embodiment shown in FIG. 10F, the plurality of elastic elements1019 are identical and spatially arranged to be each equally spacedapart. From mechanics based on Hooke's law, this serves to convertexternal vibration to translational movement of the polarizer 1012 alongthe plane 1017. In this alternative configuration, there is at least onefurther actuator 1055, 1057 with each coupled to one of the plurality ofelastic elements 1019, wherein the plurality of actuators 1015, 1055 and1057 are configured to move the polarizer 1012 along an arc 1009 thatlies in the plane 1017. In the alternative configuration shown in FIG.10F, any one of the plurality of actuators 1015, 1055 and 1057 may actas a sensor to measure the magnitude of the arc 1009, so that there is asensor coupled to at least one of the plurality of elastic elements1019. However, it will be appreciated that one or more additionalelastic elements (not shown) may serve as the one or more sensors tomeasure the arc 1009 rotation.

In the embodiment shown in FIG. 10F, the actuator 1055 is arbitrarilychosen as the sensor. This is realised by connecting the actuator 1055to an amplifier 1036 that receives an electrical signal generated whenthe actuator 1055 senses movement of the polarizer 1012 brought about byactivation of the actuators 1015 and 1057 from being driven by agenerator of driving voltage 1038, to which the actuators 1015 and 1057are coupled. The actuators 1015 and 1055 and the actuator 1055, actingas a sensor, are fixed in a rigid box 1034. From the sensor (i.e. theactuator 1055), a signal goes to a self-tuning generator 1035 comprisingthe amplifier 1036, a phase shifting circuit 1039 and the generator ofdriving voltage 1038, which goes to drive the plurality of actuators1015 and 1057 at resonance. The phase shifting circuit 1039 is tuned toachieve maximum amplitude of oscillations of the polarizer 1012 throughthe plurality of actuators 1015 and 1057. The suspension of thepolarizer 1012, using the plurality of elastic elements 1019, serves toenhance oscillations experienced by the polarizer 1012.

The results from using the third implementation of the drive mechanismare shown in FIG. 11. FIG. 11 shows a plot 1102 of the dependence oflight intensity through the modulator against angle between polarizationand polariser. The plot 1104 is the dependence of light intensitythrough the modulator against time (right). In each of the plots 1102and 1104, the reference numeral 1103 denotes the results obtained whenthe rotation stage is set to extinction, while the reference numeral1105 denotes the results obtained when the rotation stage set at anangle δ with respect to the extinction. The lock-in amplifier 1005 willgive zero output for the case 1103; but for the case 1105, it will givepositive output voltage proportional to the value of 6.

The actuator 1015 is preferably a piezo-electric strip, although abimetallic strip may also be usable. The size of the polarizer 1012 isaround 8 mm by 12 mm, the diameter of the actuator 1015 is around 13 mm.The protection box 1037 has length 1041 and breadth 1043 both of around35 mm, and height 1045 of around 16 mm. The resonance frequency isaround 242 Hz. At operating voltage 40 V peak to peak at resonance, thetilt of the polarizer 1012 is around 1 milliradian. With 3 mW laser at650 nm, using a silicon photo-detector for the detector 1006 (refer FIG.10A), a digital function generator 1007 and an analogue phase-lockamplifier 1005, measurement of polarisation rotation is achieved withsensitivity of 1 microradian.

To measure small values of rotation of light polarization, the bestsensitivity is obtained usually polarization modulation techniques. Thedetection stage 508 shown in FIG. 10A is based on a polarizationmodulation technique that is sensitive enough to measure small values ofrotation of light polarization. Further, as the detection stage 508 doesnot use any magnetically driven modulator, the detection stage 508 isnot affected by magnetic field, thereby providing an advantage oversystems which are affected by magnetic field. Such systems include aFaraday effect modulator which has light go through a paramagneticmedium in a coil where the oscillating current produces tilt ofpolarization; and an ellipsometer which uses a rotating polaroid andretarder driven by a motor which is affected by magnetic field. Thedrive mechanism of the detection stage 508 is in the millimeter rangewhich allows use in optical systems, where space is limited. Thisprovides an advantage over a photo-elastic modulator (PEM), such asModel FS50 from “Hinds International”, which has a large dimension.

The manufacture cost of the optical head provided by the componentsshown in FIGS. 10E and 10F can be kept around $40, which allows it to bemass produced, as compared to the Model FS50 from “Hinds International”,which costs around $4000. It is also possible to have the optical headused as a remote magnetic field sensor (RMFS) so that it can be attachedinstead of a Hall probe to an existing power supply. The same opticalhead can be used as a remote tensometer. In this case, the sample to bemeasured is a material with known opto-elastic constant placed, wherethe strain has to be measured. The strain induces birefringence andhence rotation of polarization. Once the rotation is calibrated versusstrain for certain sample, the strain can be measured using this remotetensometer.

The detection stage 508 shown in FIG. 10A combines three features in onedevice. It is compact, cheap and not affected by magnetic field. Itprovides a polarization modulator that can be applied in systems, wheremagneto-optical Kerr effect is used to measure magnetic properties ofmaterials or for remote detection of magnetic field.

FIG. 12A shows results of using the detection stage 508 to studymagnetic materials having magneto-optical Kerr effect. Typical value ofpolarisation rotation in one atomic layer of magnetic metal is 15microrad, so that the detection stage 508 allows measurement ofmonolayer films.

FIG. 12B shows a schematic where the detection stage 508 may be used forremote detection of magnetic field, where wired measurement is notavailable. This allows magnetic field measurement, where wires cannotreach, e.g. in harsh conditions 0 deg K to 600 deg C., or in a vacuum.Here a beam of polarized light 1204, from light generated by a lasersource 1211 passing through a polarizer 1213 and goes to a sample 1206exposed to a magnetic field 1202 (or there may be no magnetic field 1202present, but the material is magnetic; or a magnetic field sensor; bothnot shown). The sample 1206 may be made in such a way thatmagneto-optical Kerr effect produces large rotation of polarizationwhich is calibrated versus the magnetic field 1202. Reflected light 1210goes through the polarization modulation stage 1002, having thepolarizer 1012 to the detector 1006. The mean direction of the polarizer1012 is set near extinction of the original polarization. Theoscillations of the polarizer 1012 produce oscillations of the intensitygoing through; the amplitude and phase of these oscillations depend onthe rotation of polarization of incoming light. Subsequent phase lockdetection of the signal from the photo-detector 1006 (see FIG. 10A)allows measurement of the rotation of polarization. Another advantage isthat the mean direction and modulation amplitude can be setindependently.

FIG. 13 shows the results of using the detection stage 508 of FIG. 10Ato measure rotation of polarisation of light passing through a 3 mmvessel with water and after adding sugar solution in steps ofconcentration 60, 100 and 120 mg/dl. The typical value of sugarconcentration in human blood is around 70 mg/dl, which can be reliablymeasured. The transient peaks shown in FIG. 13 are due to finite time ofmixing. The polarisation rotates by the angle proportional to theproduct of sugar concentration times length of light path in thesolution.

FIG. 14A shows the results of using the detection stage 508 of FIG. 10Ausing the setup shown in FIGS. 14B and 14C to monitor sugarconcentration in a process, for example: yeast or yoghurt growth, orfermentation. It does not need consumable chemicals, only water to rinsethe filter used in the setup. The duty cycle can be e.g. 55 secmeasurement and 5 sec rinsing and calibration. FIG. 14B shows theinstantaneous monitoring of sugar and flow during measurement. FIG. 14Cshows the flow at the time of rinsing and calibration.

It will be appreciated by a person skilled in the art that numerousvariations and/or modifications may be made to the present invention asshown in the embodiments without departing from a spirit or scope of theinvention as broadly described. The embodiments are, therefore, to beconsidered in all respects to be illustrative and not restrictive.

1. An ellipsometry system for measuring any one or more of molecularbinding, adsorption and desorption on a substrate, the systemcomprising: (a) a cuvette comprising (i) a body within which a cavity isformed and an opening on the body, wherein the cavity extends into theopening through which the substrate is immersed; (ii) a window formed oneach of two oppositely located walls of the body, wherein the windowsare aligned to allow light to enter through one of the two windows toreflect off the portion of the substrate immersed in the cavity and exitthrough the other of the two windows; (iii) a channel arrangementenclosed within the body of the cuvette and comprising twonon-contiguous portions, wherein one of the two non-contiguous portionsguides fluid into the cavity and the other non-contiguous portion guidesfluid out of the cavity, wherein the cuvette further comprises a fluidinlet and a fluid outlet formed on the body, and wherein the fluid inletand the fluid outlet are in fluid communication with the channelarrangement; (b) a polarized light source disposed to provide the lightthat enters into one of the two windows on the body of the cuvette; and(c) a detection stage disposed to receive the light that exits throughthe other of the two windows on the body of the cuvette, wherein thedetection stage is configured to measure polarization rotation of thereceived light, the polarization rotation caused by any one or more ofmolecular binding, adsorption and desorption occurring on the substratesurface.
 2. (canceled)
 3. The ellipsometry system of claim 1, whereinthe channel arrangement comprises: (a) an input conduit that is coupledat one end to the fluid inlet and coupled to the cavity at the otherend; and (b) an output conduit that is coupled at one end to the fluidoutlet and coupled to the cavity at the other end.
 4. The ellipsometrysystem of claim 3 wherein the input conduit extends into the cavity bygoing around a segment of the portion of the substrate immersed in thecavity.
 5. The ellipsometry system of claim 3, wherein the input conduitcomprises two branches, wherein each of the two branches has one endthat meets at a common region for coupling to the fluid inlet and theother end is coupled to opposite corners of the cavity.
 6. Theellipsometry system of claim 3, wherein the cuvette further comprises asupport structure adapted to hold the substrate with at least a portionimmersed in the cavity.
 7. The ellipsometry system of claim 6, whereinthe support structure has a surface that receives the substrate and anopposite surface that faces the input conduit of the channelarrangement.
 8. The ellipsometry system of claim 7, wherein the supportstructure comprises a biasing element that urges the substrate againstthe receiving surface of the support structure.
 9. The ellipsometrysystem of claim 8, wherein the biasing element comprises any one or moreof a leaf, a screw and a clip.
 10. The ellipsometry system of claim 1,further comprising a pump coupled to the fluid outlet of the body of thecuvette.
 11. The ellipsometry system of claim 1, wherein the body of thecuvette further comprises a reagent inlet that extends into the channelarrangement which is in fluid communication with the fluid inlet. 12.The ellipsometry system of claim 1, wherein the opening, the fluid inletand the fluid outlet are located on different surfaces of the body ofthe cuvette. 13-20. (canceled)